Lipid Transport in Hepatocytes

Bile formation is an important function of the liver. The main physiological purposes of bile are secretion of hydrophobic remainders of xenobiotics, the secretion of cholesterol, as well as the solubilization of lipids in the intestine. Despite its importance and a wealth of research, the process of bile formation is still only incompletely understood.

The scope of this project is to provide a tool for the prediction of bile flow and bile composition that can be integrated into the metabolic model developed within HepatoSys. We are establishing a kinetic compartment model of the hepatic cholesterol and phospholipid metabolism. It contains synthesis as well as uptake and conversion to other compounds including bile salt formation and enable us to examine the flow of those compounds through the hepatocyte and the formation of the bile.

At the current state, a compartment model is under development, which reduces phenomenological detailed complicated multi-step processes into simpler reactions where necessary. A main focus is the examination of the difference in the cholesterol transport processes between normal cells and cells subject to statin treatment.

Statins are widely applied to reduce hypercholesteremia, which is a main cause of arteriosclerosis and resulting diseases like hypertension, stroke or heart failure. The primary effect of statins is the inhibition of HMG-CoA-reductase, which catalyzes the rate limiting step of the de novo cholesterol synthesis, which eventually leads to lowered cholesterol levels in blood plasma lipoproteins [1,2].

The fate of the statins is studied in the Network Detoxification of HepatoSys. The impact of reduced cholesterol levels in blood plasma to the lipoprotein composition can be studied by means of the model established by Hübner et al. [3]. This project here focuses on the so far unknown effects of statins on the composition of cellular membranes, the formation of bile salts and finally the bile composition.

Fig. 1 HepG2 cells were labeled with 2.5 μM C6-NBD-PC for 20 min on ice and subsequently washed with PBS. A fluorescence lifetime image of polarized HepG2-cells forming a bile canaliculus (arrowhead) is shown. The fluorescence lifetimes of C6-NBD-PC are strongly environment dependent ranging from about 4 ns in the lumen of the bile canaliculus (blue pseudocolor) to about 12 ns in the plasma membrane (red pseudocolor). Intermediate lifetimes are pseudocolored according to the scale. The white bar corresponds to 10 μm. Experiments were performed at 25°C.

In order to establish and verify the model, we are closely cooperating with the experimental group of Prof. Andreas Herrmann at the Institute of Biology, Humboldt Universität zu Berlin. There, fluorescence labeled lipid substrates are supplied to HepG2 cells in order to characterize their traffic within the cell and into the bile compartment [4]. In addition to the measurement of the intensity of the fluorescent marker signal, the lifetime of fluorescence is also evaluated. This enables to assess not only the distribution of the label and thereby the distribution of the according membrane compounds but also the local environment (Fig. 1) [5].

Combined measurement and model adaptation is required to satisfactory understand the flow of cholesterol and lipids through the hepatocyte into the bile. Once established, the model will help to evaluate the changes in the network on application of statins.

Researchers

Oliver Frey
Sascha Bulik
Prof. Hermann-Georg Holzhütter

in cooperation with Martin Stöckl, Molecular Biophysics Group, Prof. Andreas Herrmann, HU Berlin

This project is part of the HepatoSys Network Detoxification (Sp 6: Modeling of hepatic bile formation and biliary excretion of statins in hepatocytes).

References

[1] Endo A. (1988) Chemistry, biochemistry, and pharmacology of HMG-CoA reductase inhibitors. Klin Wochenschr., 66(10):421-7. [PubMed]

[2] Cardenas GA, Lavie CJ, Cardenas V, Milani RV, McCullough PA. (2008) The importance of recognizing and treating low levels of high-density lipoprotein cholesterol: a new era in atherosclerosis management. Rev Cardiovasc Med., 9(4):239-58. [PubMed]

[3] Hübner K, Schwager T, Winkler K, Reich JG, Holzhütter HG. (2008) Computational lipidology: predicting lipoprotein density profiles in human blood plasma. PLoS Comput Biol., 23;4(5):e1000079. [PubMed]

[4] Stöckl M, Plazzo AP, Korte T, Herrmann A. (2008) Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy of fluorescent lipid analogues. J Biol Chem., 283(45):30828-37. [PubMed]

[5] Krämer B, Koberling F, PicoQuant GmbH, Tannert A, Korte T, Hermann A. (2005) Fluorescence Lifetime Imaging (FLIM) based analysis of lipid organization in hepatocytes using the MicroTime 200. Application Note